The canonical cytosolic glutathione S-transferases (GSTs) contribute highly specific, isoform-dependent, antioxidative stress functions that modulate progression of diseases including atherosclerosis, Parkinson's disease, Alzheimer's disease, cataract formation, drug-induced hepatic toxicity, or cancer. This proposal aims to fill gaps in our understanding at the molecular level of GST-dependent oxidative stress responses and to understand the role of protein dynamics in substrate promiscuous detoxification enzymes, for which the GSTs provide an excellent model. GSTA4-4 catalyzes the conjugation of glutathione (GSH) to several lipid or prostaglandin peroxidation products of oxidative stress, such as 4-hydroxynonenal (HNE), 13-Oxo, or isoprostanoids (Isops). Although HNE and Isops may be toxic at high concentrations due to their electrophilic reactivity, they also are critical to homeostatic mechanisms wherein they regulate transcription of several stress response elements. Both HNE and isoprostanoids include chiral centers and prochiral centers leading to multiple possible diasatereomeric products of GSH conjugation. However, the stereochemical selectivity of human GSTA4-4 with respect to substrate and product has not been determined. Moreover, preliminary modeling suggests that GSTA4-4 has recruited the catalytic Tyr-9 and Arg-15 of the canonical A-class GSTs for a new purpose;these residues in GSTA4-4 may provide recognition for both HNE enantiomers, to allow substrate stereopromiscuity. Therefore, Specific Aims 1 and 2 encompass complete determination of the stereochemical course of GSTA4-4 catalyzed conjugation of GSH to HNE, as well as crystallographic analysis of the individual HNE enantiomers complexed with GSTA4-4, and in the ternary complexes with a nonreactive GSH analog. In conjunction with these aims, the stereoselectivity of the glutathione conjugate (GS-X) transporters MRP2 and RLIP76 will be determined and compared to GSTA4-4. Such a comparison explores the possibility that GSTA4-4 and transporters co-evolved with matching stereoselectivity. Specific Aim 3 explores the differential dynamics of the promiscuous archetypal detoxification enzyme GSTA1-1 with the substrate specific GSTA4-4, to which it shares a nearly identical crystal structure. This aim includes a comparison of the dynamics of GSTA1-1 and A4-4 in the presence and absence of several structurally unrelated substrates. In Specific Aim 3 mutants of GSTA1-1 and A4-4 will be made to incrementally invert their relative specificity and promiscuity, and for this series of mutants, local protein dynamics will be measured by H/D exchange mass spectrometry and fluorescence lifetime distribution analysis. These studies explore the possibility that ligand-induced conformational change, or "induced fit" can be used to achieve substrate promiscuity as well as substrate selectivity. Together, these studies add to our knowledge of GST-dependent oxidative stress responses as well as our understanding of the structure/function/dynamics of proteins that contribute to specificity vs. promiscuity.